Regulation of immune and inflammatory responses in animals is important in disease management. Immune responses can be regulated by modifying the activity of immunoregulatory molecules and immune cells. Examples of such immunoregulatory molecules include IL-18, caspase-1 and IL-12. These molecules have been found to play a role in the treatment of several disorders including allergy, cancer, and pathogenic infection.
Monocytes and macrophages represent the first line of defense against disease. Various diseases and infections activate transcriptional and posttranslational events in monocytes and macrophages, which lead to the production of cytokines such as IL-18 and IL-12. These cytokines in turn activate responses in T and B cells helping to eliminate pathogens and/or disease in a animal. Both IL-18 and IL-12 augment cellular immunity by stimulating T cells to produce interferon gamma (IFN-γ) which inhibits the production of IgE formation without compromising B cell proliferation. IL-18, formerly referred to as interferon gamma inducing factor (IGIF), stimulates T cells to produce IFN-γ and has been isolated from humans, dogs, and mice. A cDNA encoding human IL-18 was isolated and used to express recombinant human IL-18 by Ushio et al., 1996, J. Immunol. 156, 4274-4279, GENBANK® accession number D49950. Feline IL-18 cDNA has a 85.8% homology to human IL-18 cDNA and feline IL-18 protein has a 81.7% homology to human IL-18 protein. A cDNA encoding canine IL-18 was isolated and used to express recombinant canine IL-18 by Okano et al., 1999, J. Interferon Cytokine Res. 19, 27-32, GENBANK® accession number Y11133. Feline IL-18 cDNA has a 90.7% homology to canine IL-18 cDNA and feline IL-18 protein has a 88.5% homology to canine IL-18 protein. A cDNA encoding murine IL-18 was isolated and used to express recombinant murine IL-18 by Okamura et al., 1995, Nature 378, 88-91, GENBANK® accession number D49949. Feline IL-18 cDNA has a 73.8% homology to murine IL-18 cDNA and feline IL-18 protein has a 70% homology to murine IL-18 protein. A cDNA encoding rat IL-18 was isolated by Culhane, et al. Mol. Psych. 3, 362-366 (1998), GENBANK® accession number AJ222813. Feline IL-18 cDNA has a 73.4% homology with rat IL-18 cDNA, and feline IL-18 protein has a 70.7% homology with rat IL-18 protein. A cDNA encoding equine IL-18 was isolated by Nicolson, et al. (unpublished, direct submission to GENBANK®, accession number Y11131). Feline IL-18 cDNA has a 92% homology to equine IL-18 cDNA and feline IL-18 protein has a 89% homology to equine IL-18 protein. A cDNA encoding pig IL-18 was isolated by Penha-Goncalves, et al. (unpublished, direct submission to GENBANK®, accession number Y11132. Feline IL-18 cDNA has a 90.2% homology to pig IL-18 cDNA and feline IL-18 protein has a 85.9% homology to pig IL-18 protein. Expression of active IL-18 is controlled by caspase-1 (IL-1β converting enzyme). That is, IL-18 lacks a signal peptide so the precursor form of IL-18 (pro IL-18) is cleaved by caspase-1 resulting in a mature protein that is biologically active.
IL-12 is a heterodimer comprised of two subunits p40 and p35 which are covalently linked by a disulfide bond to form an active molecule. Simultaneous expression of the two subunits is necessary for the production of the biologically active heterodimer. Both human and murine p35 and p40 IL-12 single chain proteins (i.e., a single protein containing both p35 and p40 subunits) have been produced; see e.g., Lieschke et al., 1997, Nature Biotechnology 15, 35-40. Co-expression of the human and murine p35 and p40 cDNA subunits of IL-12 resulting in a biologically active IL-12 heterodimer was achieved by Gubler et al., 1991, Proc. Natl. Acad. Sci. U.S.A., 88, 4143-4147 and Schoenhaut et al., 1992, J. Immunol., 148, 3433-3440, respectively. cDNAs encoding canine IL-12 p35 and p40 subunits were isolated and co-transfected to express canine IL-12 by Okano et al., 1997, J. Interferon Cytokine Res. 17, 713-718. cDNAs encoding feline p35 and p40 subunits have been isolated and expressed; see, for example, Fehr et al., 1997, DNA Seq. 8, 77-82; Schijns et al., 1997, Immunogenetics 45, 462-463; Bush et al., 1994, Molec. Immunol. 31, 1373-1374. At the amino acid level, canine and feline IL-12 p40 subunit share 92.7 percent identity to each other; share 84.8 and 84.2 percent identity to human IL-12 p40, respectively; and share 67.4 and 68.9 percent identity to murine IL-12 p40, respectively. IL-12 shares some biological activities with IL-18 including IFN-γ production in T cells. IL-18 and IL-12 in combination work synergistically to increase IFN-γ production in T cells; as such these cytokines when utilized alone or in combination can be very effective in mediating IgE responses.
Caspase-1 may play a key role in the processing of IL-18 precursor in cells where IL-18 is produced. It may be that coexpression of caspase-1 along with IL-18 may be necessary for the proper processing of the IL-18 precursor and enhanced secretion of the processed IL-18 mature polypeptide. A cDNA encoding equine caspase-1 was isolated by Wardlow, et al. (unpublished; direct submission to GENBANK®, accession number AF090119). Feline caspase-1 cDNA has a 71% homology to equine caspase-1 cDNA and feline caspase-1 protein has a 48.8% homology to equine caspase-1 protein. A cDNA encoding equine caspase-1 was isolated by Cerretti, et al. (Science 256, p 97-100 (1992); GENBANK® accession number M87507). Feline caspase-1 cDNA has a 60% homology to human caspase-1 cDNA and feline caspase-1 protein has a 60% homology to human caspase-1 protein. A cDNA encoding rat caspase-1 (called interleukin-1 beta converting enzyme) was isolated by Keane, et al. (Cytokine 7(2) 105-110 1995); GENBANK® accession number U14647). Feline caspase-1 cDNA has a 55.4% homology to rat caspase-1 cDNA and feline caspase-1 protein has a 40.2% homology to rat caspase-1 protein. A cDNA encoding murine caspase-1 was isolated by Molineaux, et al. (Proc Natl. Acad. Sci. 90, 1809-1813, 1993); GENBANK® accession number L28095). Feline caspase-1 cDNA has a 55.7% homology to murine caspase-1 cDNA and feline caspase-1 protein has a 38.5% homology to murine caspase-1 protein. A cDNA encoding canine caspase-1 was isolated by Taylor, et al. (2000) DNA Seq. 10(6), pp 387-394; GENBANK® accession number AF135967). Feline caspase-1 cDNA has a 90% homology to canine caspase-1 cDNA.
To date, however, neither IL-18 nor caspase-1, nor the nucleic acid molecules encoding such proteins, have been isolated from cats. Neither have IL-12 single chain proteins been produced using feline or canine IL-12 subunits. As such there remains a need for compounds and methods to regulate an immune response in cats and dogs through manipulation of IL-18, caspase-1 and IL-12 single chain activities.